Cancer is a genetic disease caused by mutations acquired in somatic cells. A main driving force behind this mutation is chemical alterations in DNA resulting from cell-derived processes and exogenous DNA damaging agents. A paradox is that the repair systems that normally reverse these DNA lesions must also be responsible for transforming them into heritable sequence alterations. Thus, malignancy is a result of DNA repair failure. The double-stranded chromosome break is a DNA lesion of particular importance as it gives rise to the chromosomal rearrangements that are nearly ubiquitous in cancer. Cells possess two mechanistically distinct pathways of double-strand break repair, homology-directed repair and nonhomologous end joining (NHEJ). The long-term objective of this proposal is to understand how the balance between these two pathways is maintained and might therefore be perturbed during mutagenesis, which first requires a detailed understanding of their mechanisms. We have developed novel genetic systems specifically designed to study NHEJ in budding yeast. Their essential features are creation of a chromosome break by expressed endonucleases in such a way that simple genetic and/or physical analyses can be used to monitor break formation and subsequent repair. This proposal exploits these methodologies to perform a systematic mutational analysis of NHEJ, toward the following specific aims. In Specific Aim #1, previous work is extended to address the potential involvement of essential and redundant proteins that we hypothesize to have a high likelihood of participating in NHEJ, specifically SMC proteins, chromatin modifying complexes and checkpoint proteins. The remaining aims are a focused analysis of the multifunctional enzyme complexes with known roles in yeast NHEJ: Mrel 1/Rad50/Xrs2, Ku and DNA ligase IV. Specific Aim #2 explores the structure-function relationships of these proteins by using separation-offunction analysis to gain insight into their discrete contributions to NHEJ. Specific Aim #3 seeks to explore the sequence of events in NHEJ by using physical analysis in carefully timed break and repair assays. Specific Aim #4 seeks to identify NHEJ core complex mutants that are specifically deficient in joining incompatible DNA ends, hypothesized to be deficient in recruiting polymerases and nucleases.